Method for detecting streptococcus bacterium contained in milk

ABSTRACT

The object is to provide a lysis method and lysis treatment solution for efficiently lysing cells of various Streptococcus bacteria in milk of a livestock animal to release a specific antigen substance contained in the cells for detecting whether causative bacterium of mastitis is a Streptococcus bacterium or not by using the milk, as well as a detection method using an immunochromatographic device. There is provided a method for lysing a Streptococcus bacterium, which comprises the step of mixing a lysis agent containing a lytic enzyme with milk obtained form a livestock animal to lyse a Streptococcus bacterium existing in the milk. The lytic enzyme is preferably at least one selected from the group consisting of lysozyme, labiase, and β-N-acetylglucosaminidase.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.15/105,481 filed Jun. 16, 2016, which is the National Phase under 35U.S.C. § 371 of International Application No. PCT/JP2014/083461, filedon Dec. 17, 2014, which claims the benefit under 35 U.S.C. § 119(a) toPatent Application No. JP 2013-261825, filed in Japan on Dec. 18, 2013,all of which are hereby expressly incorporated by reference into thepresent application.

TECHNICAL FIELD

The present invention relates to a lysis method and detection method fordetecting a Streptococcus bacterium, which is a causative bacterium ofmastitis, in milk of livestock.

BACKGROUND ART

Milk of livestock animals, of which typical examples are cow, sheep, andgoat, may not be sterile, and may be contaminated with certainmicroorganisms due to diseases or environment. In particular, it isknown that animals with a disease caused by infection of a microorganismin the udder often discharge a lot of the microorganisms into milk.Typical diseases of livestock animals caused by infection of amicroorganism include mastitis.

Mastitis is inflammation of the laticifer system or milk gland tissue,and it is caused largely by invasion, colonization and proliferation ofa microorganism in the udder. Although many kinds of animals contractmastitis, it is said that, especially concerning cow's mastitis in dairycows, 15 to 40% of the whole dairy cows contract mastitis, and thus itis one of the extremely important diseases for dairy farmers. If a dairycow contracts mastitis, not only the milk synthesis function isinhibited to result in reduction of lactation amount, or even stop oflactation as the case may be, but also enormous economical losses areimposed on dairy farmers, such as cost of medical treatment and penaltyconcerning milk price due to degradation of milk quality. Furthermore,it also increases the labor of dairy farmers, since, for example,milking of teats suffering from mastitis must be separately performedfor preventing infection.

Mastitis is caused by infection of various microorganisms. Among thecausative bacteria, Streptococcus bacteria (also referred to asstreptococci) are known as frequently found causative bacteria.

As the method for detecting Streptococcus bacteria in milk,cultivation-based methods are widely used. Since the cultivation-basedmethods require several days for obtaining a result, they are notsuitable for quick identification of causative bacteria. In contrast,identification methods based on an antigen-antibody reaction using anantibody directed to an ingredient specific to a causative bacterium,especially the immunochromatographic method, can provide the result inseveral tens of minutes, and therefore they are widely used as quick andconvenient inspection methods (for example, Patent document 1). Theinventors of the present invention have examined use of animmunochromatographic method also as a method for detecting a substancecontained in milk of livestock animals (Patent document 2).

In immunological measurement methods such as the immunochromatographicmethod, when an antigen as target of the detection is an ingredientcontained in bacterial cells, it is necessary to lyse bacterial cells torelease antigens from the inside of the cells to the outside of thecells. It is known that labiase exhibits bacteriolytic activity againstStreptococcus bacteria (Non-patent document 1). However, Non-patentdocument 1 does not describe lysis effect in a high protein and high fatcontent solution such as milk.

PRIOR ART REFERENCES Patent Documents

-   Patent document 1: Japanese Patent Unexamined Publication (KOKAI)    No. 1-244370-   Patent document 2: Japanese Patent Unexamined Publication (KOKAI)    No. 2012-122921

Non-Patent Document

-   Non-patent document 1: Microbiol. Immunol., 2009, 53:45-48

SUMMARY OF THE INVENTION Object to be Achieved by the Invention

Mastitis is caused by infection of various microorganisms, and thereforeantibiotics that exhibit efficacy in therapeutic treatment therefore maydiffer depending on type of causative microorganism. Therefore, earlydiagnosis and therapeutic treatment of mastitis are very important.Further, when certain specific types of microorganisms have causedmastitis, they may transmit to other teats of the individual or otherindividuals, and therefore it is extremely important to quickly andconveniently identify the causative bacteria existing in milk.

The cultivation-based methods widely used as methods for detecting abacterium have a problem that they require several days to obtain aresult. In contrast, the immunological measurement methods based on anantigen-antibody reaction, such as the immunochromatographic method,have an advantage that they enable quick and convenient detection ofcausative bacteria, and thus enable early therapeutic treatment with anantibacterial agent. In order to highly sensitively detect a specificsubstance in cells of a causative bacterium by an immunologicalmeasurement method, when the specific substance (antigen) is anintracellular ingredient, it is necessary to highly efficiently lyse thecells to release the antigen in the inside of the cells to the outsideof the cells. However, when milk is used as a test sample, conventionaltechniques cannot provide sufficient lysis in many cases, because of theinfluences of proteins such as casein, milk fat globules, and so forthcontained in milk in large amounts. According to the investigations ofthe inventors of the present invention, it has been found thatactivities of common lytic enzymes markedly reduce in a systemcontaining milk. As described above, any effective lysis method fordetecting a Streptococcus bacterium contained in milk is not known amongthe conventional techniques.

An object of the present invention is to provide a lysis method andlysis treatment solution for efficiently lysing various Streptococcusbacteria in milk of a livestock animal to release a specific antigensubstance contained in cells for detecting whether causative bacteriumof mastitis is a Streptococcus bacterium or not by using the milk, aswell as a detection method using an immunochromatographic device.

Means for Achieving the Object

The inventors of the present invention found that Streptococcus uberiscontained in milk can be efficiently lysed by using a lysis agentcontaining at least lysozyme or labiase, and Streptococcus agalactiaecan be efficiently lysed by using a lysis agent containing a lyticenzyme selected from lysozyme, labiase, and β-N-acetylglucosaminidase.They further found that, for efficiently performing a lysis treatment ofStreptococcus bacteria contained in milk, the cells can be highlyefficiently lysed by using the labiase enzyme known to have a lysisactivity together with lysozyme further added in a specific amount, sothat the L7/L12 protein contained in the cells can be released, and suchlysis can be used for highly sensitive detection of Streptococcusbacteria, and accomplished the present invention.

The present invention thus provides the followings.

-   [1] A method for lysing a Streptococcus bacterium, which comprises:

the step of mixing a lysis agent containing at least one selected fromthe group consisting of lysozyme, labiase, and β-N-acetylglucosaminidasewith milk obtained form a livestock animal to lyse a Streptococcusbacterium existing in the milk, and wherein:

the Streptococcus bacterium is Streptococcus uberis, and the lysis agentcontains at least one of lysozyme and labiase, or

the Streptococcus bacterium is Streptococcus agalactiae, and the lysisagent contains at least one selected from the group consisting oflysozyme, labiase, and β-N-acetylglucosaminidase.

-   [2] A method for lysing a Streptococcus bacterium, which comprises    the step of mixing a lysis agent containing at least lysozyme and    labiase with milk obtained form a livestock animal to lyse the    bacterium existing in the milk.-   [3] A lysis agent for lysing a Streptococcus bacterium contained in    milk, which contains at least one selected from the group consisting    of lysozyme, labiase, and β-N-acetylglucosaminidase, or contains at    least lysozyme and labiase.-   [4] The lysis agent according to [3], which is for use in a method    for diagnosing mastitis of a livestock animal.-   [5] A method for detecting a Streptococcus bacterium contained in    milk of a livestock animal, which comprises the method according to    [1] or [2], and further comprises the step of detecting a specific    substance derived from the inside of cells and released by lysis.-   [6] The method according to [5], wherein the step of detecting a    specific substance is performed by an immunochromatographic method.

The present invention also provides the followings.

-   [1] A method for lysing a Streptococcus bacterium contained in milk,    which comprises

the step of mixing a lysis agent containing lysozyme, labiase, and anonionic surfactant with the milk to lyse the bacterium existing in themilk, or

the step of mixing a lysis agent containing at least one selected fromthe group consisting of lysozyme, labiase, and β-N-acetylglucosaminidasewith the milk to lyse the bacterium existing in the milk, wherein theStreptococcus bacterium is Streptococcus agalactiae.

-   [2] The lysis method according to [1], wherein the bacterium is    Streptococcus uberis or Streptococcus agalactiae.-   [3] The lysis method according to [1] or [2], wherein the nonionic    surfactant comprises a polyoxyethylene alkyl phenyl ether and/or a    polyoxyethylene sorbitan fatty acid ester.-   [4] The lysis method according to any one of [1] to [3], wherein, in    the step of mixing the lysis agent with the milk to lyse the    bacterium existing in the milk, final concentration of the nonionic    surfactant is not lower than 0.03% and not higher than 3%.-   [5] The lysis method according to [4], wherein, in the step of    mixing the lysis agent with the milk to lyse the bacterium existing    in the milk, final concentration of lysozyme is not lower than 0.5    mg/ml and not higher than 200 mg/ml; and/or

in the step of mixing the lysis agent with the milk to lyse thebacterium existing in the milk, final concentration of labiase is notlower than 0.05 mg/ml and not higher than 20 mg/ml.

-   [6] A method for detecting a Streptococcus bacterium contained in    milk, which comprises:

the step of mixing the lysis agent with the milk to lyse the bacteriumexisting in the milk defined in any one of [1] to [5], and furthercomprises:

the step of detecting a specific substance derived from the inside ofthe bacterium cell and released by lysis.

-   [7] The detection method according to [6], which is performed by an    immunochromatographic method.-   [8] The detection method according to [7], wherein the    immunochromatographic method comprises (1) the step of contacting    the milk containing the specific substance with a test strip having    a first part retaining a labeled first antibody directed to the    specific substance, or the specific substance that is labeled, a    second part disposed downstream from the first part, on which a    second antibody directed to the specific substance is immobilized,    and a third part disposed upstream from the first part or the second    part and having voids enabling removal of milk fat globules    contained in the milk, at the third part or a part existing upstream    therefrom, and (2) the step of flowing the milk up to the second    part or a part existing downstream therefrom to obtain a detectable    signal of the label at the second part or a part existing downstream    therefrom.-   [9] The detection method according to [8], wherein the labeled first    antibody directed to the specific substance is retained in the first    part.-   [10] The detection method according to [8] or [9], wherein the third    part is constituted by two or more kinds of members having voids    that can remove milk fat globules of different particle sizes,    respectively.-   [11] The detection method according to [10], wherein the third part    is constituted by a first member disposed downstream and a second    member disposed upstream, and retention particle size of the second    member is larger than retention particle size of the first member.-   [12] The lysis agent defined in any one of [1] to [5].-   [13] The lysis agent according to [12], which is for use in a method    of diagnosing mastitis of a livestock animal.-   [14] A kit for detecting a Streptococcus bacterium contained in    milk, which comprises:

the lysis agent according to [12] or [13], and

an immunochromatographic device for detecting a specific substancecontained in milk, which comprises a test strip having a first partretaining a labeled first antibody directed to the specific substance,or the specific substance that is labeled, a second part disposeddownstream from the first part, on which a second antibody directed tothe specific substance is immobilized, and a third part disposedupstream from the first part or the second part and having voidsenabling removal of milk fat globules contained in the milk.

Effect of the Invention

It was found that, as for lysis of Streptococcus uberis or Streptococcusagalactiae, they can be highly efficiently lysed by using a specificlytic enzyme for each bacterial species, and as for lysis ofStreptococcus bacteria, they can be highly efficiently lysed by usinglabiase and a specific amount of lysozyme in combination as lyticenzymes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows effect of a lytic enzyme in detection of a Streptococcusbacterium by enzyme immunoassay (ELISA).

FIG. 2 shows a schematic sectional view of the test strip of theimmunochromatographic device prepared in Example 3, which comprises alabeled antibody-impregnated member 1 (first part), a membrane carrier 2for chromatographic development (second part), a part 3 for capturing, amember 4 serving as both a member for sample addition and a member forremoval of fat globules (third part), a member 5 for absorption, asubstrate 6, and a member 7 for removal of fat globules (third part).

MODES FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained in more detail. Inthe present invention, when a numerical value range is represented as “Xto Y”, the range includes the values X and Y as the minimum and maximumvalues. The symbol “%” is used for indicating percent on mass basis,unless especially indicated. The expression “A and/or B” means at leastone of A and B, including the cases of referring to only A, only B, andA and B.

The present invention provides a lysis agent for lysing Streptococcusbacteria contained in milk. The lysis agent of the present inventioncontains a lytic enzyme.

[Lytic Enzyme]

Type of the lytic enzyme used in the present invention is notparticularly limited, and arbitrary two or more kinds of lytic enzymesmay also be used in combination, as required. In the present invention,it is preferable to use at least one selected from the group consistingof lysozyme, labiase, and β-N-acetylglucosaminidase. When theStreptococcus bacterium is Streptococcus uberis, the lysis agentpreferably contains at least one of lysozyme and labiase, and when theStreptococcus bacterium is Streptococcus agalactiae, the lysis agentpreferably contains at least one selected from the group consisting oflysozyme, labiase, and β-N-acetylglucosaminidase.

Labiase is prepared from culture supernatant of the Streptomycesfulvissimus TU-6 strain, and is a complex enzyme mainly consisting ofβ-N-acetyl-D-glucosaminidase and muramidase. Labiase can be preparedfrom culture supernatant of the aforementioned strain, or can also beobtained by purchasing a marketed product.

When labiase is used, especially when that described in the examplesmentioned in this specification is used, from the viewpoint ofefficiently performing lysis, final concentration thereof can bepreferably 0.05 mg/ml or higher, more preferably 0.1 mg/ml or higher,still more preferably 0.5 mg/ml or higher, and in any case, it can be 20mg/ml or lower, preferably 10 mg/ml or lower, more preferably 5 mg/ml orlower. Alternatively, the final concentration thereof can be 0.0005unit/ml or higher, preferably 0.001 unit/ml or higher, more preferably0.005 unit/ml or higher, and in any case, it can be 0.2 unit/ml orlower, preferably 0.1 unit/ml or lower, more preferably 0.05 unit/ml orlower. When the term enzyme unit (unit) is mentioned for labiase in thisspecification, it refers to a value of the β-N-acetyl-D-glucosaminidaseactivity, and the enzymatic activity that releases 1 μmol ofp-nitrophenol in 1 minute when the enzyme is allowed to act onp-nitrophenyl-β-N-acetyl-D-glucosamine as a substrate is defined as 1unit, unless especially indicated.

Lysozyme is a protein consisting of a single peptide of 14.6 kDa, andcan lyse cells of bacteria by cleaving the β(1-4) glycosidic linkagebetween N-acetylmuramic acid and N-acetylglucosamine in thepeptidoglycan layer. Lysozyme can be obtained by purchasing a marketedproduct.

When lysozyme is used, especially when that described in the examplesmentioned in this specification is used, from the viewpoint ofefficiently performing lysis, final concentration thereof can bepreferably 0.5 mg/ml or higher, more preferably 1 mg/ml or higher, stillmore preferably 5 mg/ml or higher, and in any case, it can be 200 mg/mlor lower, preferably 100 mg/ml or lower, more preferably 50 mg/ml orlower. Alternatively, the final concentration of lysozyme can bepreferably 0.4 mg/ml or higher, more preferably 0.8 mg/ml or higher,still more preferably 4 mg/ml or higher, and in any case, it can be 160mg/ml or lower, preferably 80 mg/ml or lower, more preferably 40 mg/mlor lower. When amount as lysozyme is referred to in this specificationfor lysozyme, it is a value determined on the basis of decrease ofabsorbance (640 nm) observed when it is allowed to act on a suspensionof dry cells of Micrococcus Lysodeikticus in a phosphate buffer as asubstrate, unless especially indicated.

β-N-Acetylglucosaminidase is also called β-N-acetylhexosaminidase, andis an enzyme that releases terminal β-linked N-acetylglucosamine andN-acetylgalactosamine from various substrates. β-N-Acetylglucosaminidasecan be obtained by purchasing a marketed product.

When β-N-acetylglucosaminidase is used, especially when that describedin the examples mentioned in this specification is used, from theviewpoint of efficiently performing lysis, final concentration thereofcan be preferably 0.5 mg/ml or higher, more preferably 1 mg/ml orhigher, still more preferably 5 mg/ml or higher, and in any case, it canbe 200 mg/ml or lower, preferably 100 mg/ml or lower, more preferably 50mg/ml or lower. Alternatively, the final concentration thereof can bepreferably 0.05 unit/ml or higher, more preferably 0.1 unit/ml orhigher, still more preferably 0.5 unit/ml or higher, and in any case, itcan be 200 units/ml or lower, preferably 100 units/ml or lower, morepreferably 50 unit/ml or lower. When the term enzyme unit (unit) ismentioned for β-N-acetylglucosaminidase in this specification, theenzymatic activity that releases 1 μmol of p-nitrophenol in 1 minutewhen the enzyme is allowed to act onp-nitrophenyl-β-N-acetyl-D-glucosamine as a substrate is defined as 1unit, unless especially indicated.

According to the present invention, by further supplementarily addingthe enzyme lysozyme to milk containing Streptococcus bacteria inaddition to the enzyme labiase, highly efficient lysis effect can beattained, which effect can be applied to a wide range of Streptococcusbacteria. When the lysozyme and labiase described in the section ofexamples of this specification are used, from the viewpoint ofefficiently performing lysis, amount of lysozyme to be supplementarilyadded is preferably 3 mg/ml or larger, more preferably 5 mg/ml orlarger, further preferably 7 mg/ml or larger, still further preferably 9mg/ml or larger, and preferably 100 mg/ml or smaller, more preferably 50mg/ml or smaller, further preferably 20 mg/ml or smaller, per 1 mg/ml oflabiase. Alternatively, amount of lysozyme to be supplementarily addedis preferably 2.4 mg/ml or larger, more preferably 4 mg/ml or larger,further preferably 5.6 mg/ml or larger, still further preferably 7.2mg/ml or larger, and in any case, preferably 80 mg/ml or smaller, morepreferably 40 mg/ml or smaller, further preferably 16 mg/ml or smaller,per 0.1 unit/ml of labiase.

[Surfactant]

A surfactant that ionizes and becomes an ion, when it is dissolved inwater, is referred to as ionic surfactant, and a surfactant that doesnot become an ion is referred to as non-ionic (nonionic) surfactant.Ionic surfactant is further classified into anionic surfactant, cationicsurfactant, and ampholytic surfactant.

It can be expected that, by adding a surfactant to the lysis agent inaddition to the lytic enzyme, the lysis effect thereof can be enhanced.As the surfactant, any of nonionic surfactant, cationic surfactant,anionic surfactant, and ampholytic surfactant can be independently used,or any of these may be used in combination.

The lysis agent of the present invention can contain a nonionicsurfactant. It is considered that coexistence of a nonionic surfactantis particularly preferred, when the obtained cell lysate is used in animmunochromatographic method. It is considered that, as the nonionicsurfactant, any of those of ester ether type, ester type, and ether typecan be preferably used. More specifically, examples includepolyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, fattyacid sorbitan esters, alkyl polyglucosides, fatty acid diethanolamides,alkyl monoglyceryl ethers, polysorbates (formed by condensation ofseveral tens of molecules of ethylene oxide to a fatty acid sorbitanester), and so forth, but it is not particularly limited. Especiallypreferred examples include polysorbates, more specifically,polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (60) sorbitanmonostearate, polyoxyethylene (65) sorbitan tristearate, andpolyoxyethylene (80) sorbitan monooleate, and so forth, as well aspolyoxyethylene alkyl phenyl ethers, more specifically, polyoxyethylene(10) octyl phenyl ether, and so forth.

Content of the nonionic surfactant in the lysis agent (when two or morekinds of nonionic surfactants are used, it is the content as the totalamount of the nonionic surfactants) is not particularly limited so longas, for example, when an immunochromatographic method is used, flow of adeveloping solution is secured. However, in any case, as for the lowerlimit thereof, the content can be determined so that the finalconcentration of the nonionic surfactant in the mixture with milkbecomes 0.03% or higher, preferably 0.05% or higher, more preferably0.075% or higher, further preferably 0.1% or higher, particularlypreferably 0.3% or higher. As for the upper limit of the content of thenonionic surfactant, the content can be determined so that the reactioncatalyzed by the lytic enzyme and antigen-antibody reactions are notsignificantly inhibited, and in any case, it can be 10% or lower,preferably 7.5% or lower, more preferably 5.0% or lower, still morepreferably 3% or lower, particularly preferably 2% or lower.

As the anionic surfactant, any of carboxylic acid salts, sulfonic acidsalts, sulfuric acid ester salts, and so forth can be preferably used.More specific examples include alkyl ether carboxylates, linearalkylbenzenesulfonates (LAS), α-olefinsulfonates (AOS),dialkylsulfosuccinates, formaldehyde condensates ofnaphthalenesulfonate, alkyl sulfuric acid ester salts (AS),polyoxyethylenealkylsulfuric acid ester salts (AES) obtained by additionof ethylene oxide to a higher alcohol and following sulfation,phosphoric acid ester salts of a higher alcohol or ethylene oxide adductthereof, and so forth. Further specific examples include sodiumalkylsulfates such as sodium dodecylsulfate and sodium myristylsulfate,sodium N-acylsarcosinates such as sodium N-lauroylsarcosinate and sodiumN-myristoylsarcosinate, sodium dodecylbenzenesulfonate, hydrogenatedcoconut fatty acid monoglyceride monosodium sulfate, sodiumlaurylsulfoacetate, N-acylglutamates such as sodiumN-palmitoylglutamate, N-methyl-N-acylalanine sodium, and sodiumα-olefinsulfonates.

As the cationic surfactant, any of those of amine salt type andquaternary ammonium salt type can be preferably used. More specificexamples include distearyldimethylbenzylammonium chloride, benzalkoniumchloride, hexadecyltrimethylammonium bromide, hexadecyltrimethylammoniumbromide, myristyltrimethylammonium bromide, and so forth.

Examples of the ampholytic surfactant include those of amino acid type(alkylaminofatty acid salts), betaine type (alkyl betaines), amine oxidetype (alkylamine oxides), and so forth, but it is not particularlylimited. More specific examples includedimethylammoniopropanesulfonates, dodecyldimethylammoniobutyrates,lauryl betaine, and amidopropyl betaine. Further specific examplesinclude n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,n-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, andN-dodecyl-N,N-(dimethylammonio)butyrate.

[Example of Composition]

In a particularly preferred embodiment, the lysis agent compriseslysozyme, labiase, and a nonionic surfactant. Such a lysis agent iseffective for various Streptococcus bacteria, and it is especiallyeffective for Streptococcus uberis or Streptococcus agalactiae. For thelysis agent containing lysozyme, labiase, and a nonionic surfactant,particularly preferred examples of the nonionic surfactant include apolyoxyethylene alkyl phenyl ether, and/or a polyoxyethylene sorbitanfatty acid ester, and more preferred examples include a polyoxyethylenealkyl phenyl ether and a polyoxyethylene sorbitan fatty acid ester.Concentrations of such ingredients of the lysis agent can be determinedso that, in the step of lysing the bacteria existing in milk by mixingthe lysis agent with the milk, the final concentration of lysozyme isnot lower than 0.6 mg/ml and not higher than 50 mg/ml, the finalconcentration of labiase is not lower than 0.05 mg/ml and not higherthan 20 mg/ml, and the final concentration of the nonionic surfactant isnot lower than 0.03% and not higher than 3%. With a more preferredembodiment of the lysis agent containing lysozyme, labiase, and anonionic surfactant, in the step of lysing bacteria existing in milk bymixing the lysis agent with the milk, the final concentration oflysozyme is not lower than 0.5 mg/ml and not higher than 50 mg/ml, thefinal concentration of labiase is not lower than 0.1 mg/ml and nothigher than 10 mg/ml, and the final concentration of the nonionicsurfactant is not lower than 0.10% and not higher than 2.6%.

[Other Ingredients]

The lysis agent of the present invention may contain, besides the lyticenzyme and the surfactant, one or more kinds of other ingredients, solong as the intended effect is not markedly degraded. Preferred examplesof the ingredients other than the lytic enzyme and the surfactantinclude a substance that has an effect of promoting the lysis. Specificexamples include glutaraldehyde, halogen compounds, chlorhexidine,alcohols (for example, methanol, ethanol, 1-propanol, 2-propanol,1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol), phenol,hydrogen peroxide, acrinol, guanidine and salt thereof, chelatingagents, organic acids (e.g., decanoic acid) and salts thereof,polyhydric alcohols (for example, ethylene glycol, propylene glycol,diethylene glycol, glycerin, and monocaprylin), and reducing agents suchas 2-mercaptoethanol, dithiothreitol, cystine, and thiophenol, but theyare is not limited to these.

For example, when either one of guanidine and a salt thereof (forexample, guanidine thiocyanate) is used, as for the lower limit of thefinal concentration thereof at the time of mixing with milk, theconcentration (when two or more kinds of the substances are used, it isthe concentration as the total concentration of them) can be 0.1 mM orhigher, preferably 1 mM or higher, more preferably 1 mM or higher. Inany case, as for the upper limit of the final concentration at the timeof mixing with milk, the concentration can be 500 mM or lower,preferably 200 mM or lower, more preferably 100 mM or lower.

Examples of the chelating agent include ethylenediaminetetraacetic acid(EDTA) and a salt thereof, glycol ether-diaminetetraacetic acid (EGTA)and a salt thereof, polyphosphoric acid and a salt thereof, as well asmetaphosphoric acid and a salt thereof. As for the lower limit of thefinal concentration thereof at the time of mixing with milk, theconcentration (when two or more kinds of the chelating agents are used,it is the concentration as the total concentration of them) can be 0.001mM or higher, preferably 0.01 mM or higher, more preferably 0.1 mM orhigher. In any case, as for the upper limit of the final concentrationat the time of mixing with milk, the concentration can be 100 mM orlower, preferably 10 mM or lower, more preferably 5 mM or lower, fromthe viewpoint of suppressing inhibition of an antigen-antibody reaction.

[Lysis Conditions and Lysis Ratio]

In the present invention, the lysis agent can be used by mixing it withmilk. Mixing ratio of milk and the lysis agent is not particularlylimited, so long as the final concentrations of the lytic enzyme etc.are properly maintained, and sufficient lysis ratio can be secured. Ifthe lysis agent is used in a volume relatively small with respect tomilk, the milk is not diluted. Therefore, it can be expected that cellscan be detected with higher sensitivity. When the lysis agent is used ina volume relatively large with respect to milk, influences of fatglobules and proteins contained in milk are reduced, and therefore itcan be expected that cells can be detected in a shorter time. From theviewpoint that a higher ratio of milk in the mixture of milk and thelysis agent (milk/(milk+lysis agent)×100) can provide higher detectionsensitivity, the ratio can be, for example, 5% or higher, preferably 10%or higher, more preferably 20% or higher, further preferably 30% orhigher, irrespective of the other conditions. As for the upper limit ofthe ratio, if the lysis agent solidified by drying or the like is used,the ratio of milk can be made to be 100%, irrespective of the otherconditions. The ratio of the milk can be 90% or lower, 80% or lower, 70%or lower, 60% or lower, or 50% or lower, irrespective of the otherconditions. As for the upper limit, the ratio may be determined inconsideration of ease of mixing, stability of the lysis agent as asolution, and so forth.

In the present invention, it is sufficient to simply mix milk with thelysis agent. Temperature at the time of mixing, and treatment time forallowing the enzyme to act following the mixing are not particularlylimited, so long as the lytic enzyme used can exhibit the activity, andthe temperature may usually be room temperature. The treatment time forallowing the reaction after the mixing can be appropriately determinedby those skilled in the art in consideration of lysis ratio. In thepresent invention, since combination and concentrations of lytic enzymesappropriate for Streptococcus bacteria are used, the treatment time canbe shortened compared with that obtainable by use of a usual lysismethod. In the present invention, in any case, the treatment time isgenerally about several tens of minutes to several hours, and morespecifically, it can be 240 minutes or shorter, preferably 150 minutesor shorter, more preferably 90 minutes or shorter, still more preferably60 minutes or shorter, further preferably 45 minutes or shorter, stillfurther preferably 25 minutes or shorter, still more further preferably20 minutes or shorter, particularly preferably 15 minutes or shorter.The treatment time can also be made to be substantially 0 (after milkand the lysis agent are mixed, the mixture is immediately subjected tothe measurement). Temperature can be, in any case, 2 to 50° C., forexample, 35 to 40° C., and it can be determined as required inconsideration of type of the enzyme and treatment time to be used. Thetreatment can be performed in a stationary state, or with stirring.According to the present invention, even with such a short period oftime, required lysis can be attained, and detection of Streptococcusbacteria can be enabled. In the present invention, a surfactant can alsobe used in combination with the lytic enzyme. In such a case, thetreatment time may be shortened compared with the case of using only alytic enzyme, or the lysis ratio may be increased even with the sametreatment time. The treatment time is generally about several tens ofminutes to several hours, and more specifically, it can be 240 minutesor shorter, preferably 150 minutes or shorter, more preferably 90minutes or shorter, still more preferably 60 minutes or shorter, furtherpreferably 45 minutes or shorter, still further preferably 25 minutes orshorter, still more further preferably 20 minutes or shorter,particularly preferably 15 minutes or shorter. The treatment time canalso be made to be substantially 0 (after milk and the lysis agent aremixed, the mixture is immediately subjected to the measurement). Alsowhen a surfactant is used in combination, temperature can be, in anycase, 2 to 50° C., for example, 35 to 40° C., and it can be determinedas required in consideration of type of the enzyme and treatment time tobe used. Also in this case, the treatment can be performed in astationary state, or with stirring.

As for the lysis ratio referred to in the present invention, lysis ratioobserved for a suspension of a target Streptococcus bacterium subjectedto a preliminary treatment with a nonionic surfactant of a properconcentration, then further subjected to ultrasonication, and asufficient enzyme treatment, as required, can be defined as 100%, unlessespecially indicated.

[Detection Means]

Streptococcus bacteria lysed according to the present invention can bedetected by various kinds of immunological methods using an ingredientof bacterial cells as an antigen. For example, an antigen consisting ofthe ribosomal L7/L12 protein of a Streptococcus bacterium can bedetected by an immunochromatographic method, or the aforementionedantigen can also be detected by another immunological measurement methodinstead of the immunochromatographic method. Examples of immunologicalmeasurement method other than the immunochromatographic method include,for example, agglutination reaction method, enzyme immunoassay (ELISA),radioimmunoassay (RIA), fluoroimmunoassay (FIA), and so forth, but it isnot limited to these.

At the time of performing the immunological methods, a substance forblocking for preventing non-specific adsorption, or a substance forpreventing cross-reaction with bacteria other than the target bacteriummay be used. In particular, in order to prevent reaction of the antibodyused and the protein A of Staphylococcus aureus, globulin that does notparticipate in the reaction with the antigen can be added. When globulinis used, it can be added in an amount of 0.01 μg/ml or larger,preferably 0.1 μg/ml or larger, more preferably 1 μg/ml or larger, asthe final concentration at the time of the reaction, from the viewpointof suppressing false positive results. Further, in any case, from theviewpoint of not inhibiting the objective antigen-antibody reaction, itcan be used in an amount of 10 mg/ml or smaller, preferably 5 mg/ml orsmaller, more preferably 1 mg/ml or smaller. Such conditions concerningthe final concentration are especially suitable for theimmunochromatographic method.

When an immunochromatographic method is used, it can be carried outtypically as follows.

[Immunochromatographic Method and Immunochromatographic Device]

An antigen-antibody reaction can be detected by the sandwich assay usinga “labeled first antibody directed to a specific substance” retained bya first part, and a “second antibody directed to the specific substance”immobilized on a second part. Alternatively, an antigen-antibodyreaction may also be detected by the competition method using a labeledspecific substance retained by a first part, and an antibody directed tothe specific substance immobilized on a second part. However, in thepresent invention, the sandwich assay method is preferred, since itshows high detection sensitivity and gives a line indicating detectionof antibody as a positive result.

The immunochromatographic device is a device for detecting a specificsubstance contained in milk by an immunochromatographic method, whichcomprises a test strip having a first part retaining a labeled firstantibody directed to the specific substance, or the specific substancethat is labeled, a second part disposed downstream from the first part,on which a second antibody directed to the specific substance isimmobilized, and a third part disposed upstream from the first part orthe second part and having voids enabling removal of milk fat globulescontained in the milk. As a specific example of the structure of thetest strip, that of the test strip of which schematic sectional view isshown in FIG. 2 can be mentioned. In FIG. 2, a member 7 for removal offat globules (third part) is disposed downstream from a member 4 forsample addition, and upstream from a labeled antibody-impregnated member(first part) 1.

The immunochromatographic device can be produced in a known manner byusing marketed materials.

The material used for the first part is not particularly limited, solong as a material enabling immunochromatography is chosen, butpreferred examples include a fiber matrix of cellulose derivative etc.,filter paper, glass fiber, cloth, cotton, and so forth.

The material used for the second part is not particularly limited, solong as a material enabling immunochromatography is chosen, butpreferred examples include cellulose nitrate, mixed cellulose nitrateester, polyvinylidene fluoride, nylon, and so forth.

The material used for the third part preferably has voids that enableremoval of milk fat globules contained in milk and having a diameter ofabout 1 to ten and several micrometers. The third part must be disposedupstream from the aforementioned second part consisting of a porousmembrane having a pore diameter of several tens to several hundreds nm,and is preferably disposed upstream from the aforementioned first part,i.e., at a position at which a sample solution first contacts with andpasses through the test strip.

The voids of the third part may have a size that enables removal of milkfat globules, and retention particle size is preferably 0.1 to 10 μm,more preferably 1 to 3.5 μm. The material is not particularly limited,so long as a material having voids showing a retention particle sizewithin the aforementioned range is chosen, but preferred examplesinclude a matrix of fibers such as cellulose derivatives, filter paper,glass fiber, cloth, cotton, and so forth. The retention particle sizemeans such a particle size of milk fat globules that milk fat globuleshaving a particle size not smaller than the retention particle sizecannot pass through the voids and retained by the third part, andsubstantially corresponds to average pore size of the voids of the thirdpart, and 50% or more, preferably 60% or more, more preferably 70% ormore, still more preferably 80% or more, particularly preferably 90% ormore, most preferably 98% or more, of milk fat globules having aparticle size not smaller than the retention particle size cannot passthrough the voids and are retained by the third part. Ratio of milk fatglobules to be retained can be measured by a method well known to thoseskilled in the art. For example, the catalogue of GF/B provided by GEHealthcare Bioscience describes that the retention particle size(particle diameter for which retention efficiency is 98%, the termretention particle size used in this specification has this meaning,unless especially indicated) thereof is 1.0 μm, and such a particle sizeas mentioned above can be confirmed by a method well known to thoseskilled in the art.

The aforementioned third part may consist of a single kind of materialhaving a specific retention particle size, or may consist of a laminatecomprising materials having different retention particle sizes andintegrally adhered so that the retention particle size becomes smallerstepwise, in order to increase milk fat globule separation efficiency.Such a third part as mentioned above constituted by two or more kinds ofmembers that can remove milk fat globules of different particle sizesconstitutes a preferred embodiment of the present invention, and in amore preferred embodiment of the present invention, the third part isconstituted with a first member disposed downstream and a second memberdisposed upstream, and the retention particle size of the second memberis larger than the retention particle size of the first member. When thethird part is constituted with such two kinds of members, it ispreferred that the retention particle size of the first member disposeddownstream is 1.0 to 2.0 μm, and the retention particle size of thesecond member disposed upstream is 3.0 to 3.5 μm. In order to highlysensitively detect a specific substance from milk containing milk fatglobules of high concentration and wide particle size distribution,especially such milk not diluted after milking, it is preferred that thethird part is constituted with a combination of a member having a smallretention particle size and a member having a large retention particlesize.

The aforementioned first part retains a labeled first antibody directedto a specific substance, or a labeled specific substance. If the firstpart retains a labeled first antibody directed to a specific substance,the specific substance can be detected by the sandwich assay method. Ifthe first part retains a labeled specific substance, the specificsubstance can be detected by the competition method. Since the sandwichassay method that shows high detection sensitivity and gives a lineindicating detection of antibody as a positive result is more preferredfor the present invention, the first part preferably retains a labeledfirst antibody directed to a specific substance.

When the first part is made to retain a labeled first antibody directedto a specific substance, two kinds of antibodies, the first antibodydirected to the specific substance, and a second antibody also directedto the specific substance, are used. In order to enable detection of thespecific substance by the sandwich assay method, the aforementionedfirst antibody and second antibody are antibodies that cansimultaneously bind to the specific substance, and it is preferred thatthe epitope of the specific substance to be recognized by theaforementioned first antibody is different from the epitope of thespecific substance to be recognized by the aforementioned secondantibody.

In the present invention, in order to obtain a detectable signal, thefirst antibody or the specific substance retained by the first part islabeled. Examples of the label used for the present invention include acolored particle, enzyme, radioisotope, and so forth, and it ispreferable to use a colored particle that can be visually detectedwithout any special equipment. Examples of the colored particle includemetal microparticles such as those of gold and platinum, nonmetallicparticles, latex particles, and so forth, but are not limited to these.The colored particle may have any size so long as the colored particlehas such a size that it can be transported downstream through the insideof the voids of the test strip, but it preferably has a size of 1 nm to10 μm, more preferably 5 nm to 1 μm, still more preferably 10 to 100 nm,in diameter.

[Object of Detection]

According to the present invention, a Streptococcus bacterium containedin milk can be detected. The “Streptococcus bacterium” referred to inthe present invention may be Streptococcus uberis, Streptococcusagalactiae, Streptococcus dysgalactiae, Streptococcus equinus,Streptococcus canis, Streptococcus bovis, Streptococcus pluranimalium,Streptococcus parauberis, Streptococcus mutans, Streptococcusacidominimus, or the like, unless especially indicated. According to thepresent invention, Streptococcus bacteria contained in milk obtainedfrom a livestock animal such as cow as it is, which contains fatglobules and proteins of high concentration, can be detected by usingthe lysis agent containing appropriately formulated lytic enzyme andsurfactant.

The specific substance measured in the present invention may be anysubstance so long as it is a substance that can be measured by animmunological method such as immunochromatographic method, but it ispreferably a component of a bacterium or a substance that is secreted bya bacterium. The specific substance is more preferably the L7/L12ribosomal protein of a bacterium. High detection sensitivity can beobtained for the L7/L12 ribosomal protein, since it exists in cells in alarge copy number.

[Antibody]

The antibody used in the present invention can be prepared by the methoddescribed in International Patent Publication WO00/06603. When thebacterial ribosomal protein L7/L12 is used as the antigen, the antibodycan be prepared by using a full length protein or a partial peptide ofthe bacterial ribosomal protein L7/L12 as an antigen, but it ispreferably prepared by using the full length protein as an antigen. Anantiserum containing an antibody (polyclonal antibody) that recognizesthe L7/L12 ribosomal protein can be obtained by inoculating such apartial peptide or full length protein as mentioned above as it is orcrosslinked with a carrier protein to an animal, together with anadjuvant as required, and collecting the serum of the animal. Further,the antibody purified from the antiserum can also be used. Examples ofthe animal used for the inoculation include sheep, horse, goat, rabbit,mouse, rat, and so forth, and sheep, rabbit, and so forth are especiallypreferred for preparing polyclonal antibodies. Further, it is morepreferable to use, as the antibody, a monoclonal antibody obtained by aknown method in which a hybridoma cell is prepared, and in such a case,mouse is preferred as the animal. If, as such a monoclonal antibody, amonoclonal antibody that reacts with the ribosomal protein L7/L12 of aspecific bacterium that causes mastitis, but does not react with theribosomal protein L7/L12 of a bacterium that causes mastitis other thanthe above specific bacterium is retrieved by screening, it can beutilized for diagnosing whether an animal suffers from infection by thebacterium or not.

A monoclonal antibody that recognizes a substance other than theribosomal protein L7/L12 as an antigen may also be used, so long as theantibody is a monoclonal antibody that reacts with a component of aspecific bacterium that causes mastitis or a substance secreted by sucha bacterium, but does not react with a component of a bacterium thatcauses mastitis other than the foregoing specific bacterium or asubstance secreted by such a bacterium.

Further, as the monoclonal antibody, it is preferable to use amonoclonal antibody of which antigen-antibody reaction is not inhibitedby any contaminants other than the specific substance contained in milk.For example, milk contains a large amount of proteins such as casein,and they may inhibit the reaction of the specific substance and themonoclonal antibody. When a monoclonal antibody directed to the specificsubstance is prepared in a conventional manner, for example, amonoclonal antibody of which antigen-antibody reaction is not inhibitedby casein or the like, or a monoclonal antibody of whichantigen-antibody reaction is hardly affected by casein or the like maybe preferably chosen and used. Such a monoclonal antibody can be easilyobtained by preparing monoclonal antibodies that specifically react withan antigen in a usual manner, and then selecting a monoclonal antibodyof which antigen-antibody reaction is not substantially inhibited by acontaminant such as casein by examining whether the antigen-antibodyreaction is inhibited or not in the presence of the contaminant.

EXAMPLES Example 1 (1) Preparation of Ribosomal Protein L7/L12 Antibody

According to the method described in the examples of InternationalPatent Publication WO00/06603, the L7/L12 ribosomal protein wasobtained, and monoclonal antibodies were prepared by using this protein.Among the monoclonal antibodies, a combination of two kinds ofmonoclonal antibodies that can simultaneously bind to different sites ofthe aforementioned L7/L12 ribosomal protein was selected.

(2) Evaluation of Lytic Activity by ELISA

S. uberis or S. agalactiae cell count was adjusted to 1×10⁹ (cells/ml)with physiological saline to prepare a cell suspension. Further, 6 kindsof the enzymes shown in Table 1 were each diluted with a 1:1 (v/v)mixture of milk and each buffer to a concentration of 1 mg/ml or 10mg/ml to prepare enzyme solutions.

TABLE 1 Concentration of enzyme solution (final concen- Manufac- trationafter mixing Lytic enzyme turer Buffer with cell suspension) Labiase(Lot, Ozeki Citrate/ 1 mg/ml BDBAC12)*1 phosphate (0.5 mg/ml)  Na, pH 4Lysozyme *2 Wako Pure Citrate/ 10 mg/ml  Chemical phosphate (5 mg/ml)Industries Na, pH 4 Lysostaphin Wako Pure Tris-HCl, 10 mg/ml  ChemicalpH 8 (5 mg/ml) Industries Achromopeptidase Wako Pure Tris-HCl, 10 mg/ml Chemical pH 8 (5 mg/ml) Industries β-N-Acetyl- Sigma- Citrate/ 10 mg/ml glucosaminidase *3 Aldrich phosphate (5 mg/ml) Na, pH 4 Proteinase KSigma- Tris-HCl, 0.1 mg/ml  Aldrich pH 8 (0.05 mg/ml)  *1β-N-Acetyl-D-glucosaminidase activity, 19.13 U/g-powder; Protein,24.00 mg-protein/g-powder; Lysis of lactic acid bacterium cells with0.5% (w/v) labiase *2: Titer, 0.8 mg lysozyme/mg or higher (Wako PureChemical Industries Product Standard) *3: >80 units/mg protein

As a positive control, a disrupted cell suspension obtained by stirringS. uberis or S. agalactiae cells overnight in the MOPSO buffer (0.1 M)containing 1% nonionic surfactant Triton X-100, and then ultrasonicatingthe cell suspension (output 10%, 1 minute×10 times) was used.

The monoclonal antibody 1 (10 μg/ml) in 0.05% NaN₃/PBS (50 μl) was putinto each well of a 96-well ELISA plate (Maxsorp ELISA Plate, Nunc), andadsorption of the antibody was allowed overnight at 4° C. After thesupernatant was removed, a 1% bovine serum albumin solution (in PBS, 200μl) was added, and the reaction was allowed at room temperature for 1hour to attain blocking. After the supernatant was removed, each wellwas washed several times with a washing solution (0.05% Tween 20, PBS).

The bacterial cell suspension and the enzyme solution prepared abovewere mixed at a volume ratio of 1:1, treated for 1 hour in an incubatorat 37° C., and diluted 100 times with 0.05% Tween 20 in PBS, the dilutedsuspension (50 μl) was added to each well of the plate, and the reactionwas allowed at 37° C. for 1 hour. After the supernatant was removed,wells were washed several times with the washing solution, 50 μl of themonoclonal antibody-2 (1 μg/ml, 0.05% Tween 20 in PBS) labeled withperoxidase was added, and the reaction was allowed at room temperaturefor 1 hour. After the supernatant was removed, wells were further washedseveral times with the washing solution, then a TMB solution (100 μl,KPL) was added to each well, and the reaction was allowed at roomtemperature for 10 minutes. Then, 1 mol/l hydrochloric acid (100 μl) wasadded to terminate the reaction, and absorbance was measured at 450 nm.

Lysis ratio was calculated for each enzyme from a ratio of theabsorbance to that of the positive control, of which lysis ratio wastaken as 100%. A lysis ratio of 5% or higher is indicated with +, andthat lower than 5% is indicated with −. The results are shown in Table2.

TABLE 2 Lytic enzyme S. uberis S. agalactiae Lysozyme + + Labiase + +Lysostaphin − − Achromopeptidase − − β-N-Acetylglucosaminidase − +Proteinase K − −

Each of lysozyme and labiase brought about the effect by itself on boththe bacteria. β-N-Acetylglucosaminidase brought about the effect byitself on S. agalactiae.

Example 2

The bacteria were treated in the same manner as described above withlabiase and lysozyme at final concentrations of 0.5 mg/ml and 4.5 mg/ml,respectively, adjusted with a mixture of a citrate-phosphate Na bufferand milk (mixing ratio in volume was 1:1). The results for lysis ratiocalculated from the results of ELISA are shown in FIG. 1. It wasrevealed that when lysozyme or labiase was used alone, the bacteriolyticactivity was insufficient for either the bacterium S. uberis or S.agalactiae, but if they are used in combination as a mixture in certainconcentration ranges thereof, both the bacteria can be highlyefficiently lysed.

Example 3 Preparation of Immunochromatographic Device

An immunochromatographic device was prepared as follows.

(a) Gold Colloid-Labeled Antibody-Impregnated Member

A gold colloid solution (particle size 60 nm, 0.9 mL, BB International)was mixed with 0.1 M potassium phosphate, pH 7.0, a monoclonal antibody1 (100 μg/mL) to be labeled with gold colloid was added to the mixture,and the resulting mixture was left standing at room temperature for 10minutes so that the antibody bound to the gold colloid particlesurfaces. Then, a 10% aqueous solution of bovine serum albumin (BSA) wasadded at a final concentration of 1% in the gold colloid solution, sothat the remaining surfaces of the gold colloid particles were blockedwith BSA, to prepare a solution of the monoclonal antibody 1 labeledwith gold colloid (henceforth referred to as “gold colloid-labeledantibody”). This solution was centrifuged (at 15000 rpm for 5 minutes)to precipitate the gold colloid-labeled antibody, and the supernatantwas removed to obtain the gold colloid-labeled antibody. This goldcolloid-labeled antibody was suspended in 20 mM Tris-hydrochloric acidbuffer (pH 7) containing 0.25% BSA, 2.5% sucrose, and 35 mM NaCl toobtain a gold colloid-labeled antibody solution. A glass fiber pad of astrip-like shape (10 mm×300 mm) was impregnated with the goldcolloid-labeled antibody solution (2 mL), and dried at room temperatureunder reduced pressure to obtain a gold colloid-labeledantibody-impregnated member 1 (first part).

(b) Part for Capturing Complex of Antigen and Gold Colloid-LabeledAntibody

A nitrocellulose membrane having a width of 25 mm and a length of 300 mmwas prepared as a membrane carrier 2 for chromatographic developmentwith chromatography medium (second part).

A solution containing the monoclonal antibody 2 (1.5 mg/mL) was appliedin the shape of a line in a volume of 1 μL/cm to the membrane carrier 2for chromatographic development at a position of 10 mm from the end onthe side of the chromatography development starting point, and dried at50° C. for 30 minutes, and then the membrane carrier was immersed in a0.5% sucrose solution for 30 minutes, and dried overnight at roomtemperature to obtain a part 3 for capturing the complex of theribosomal protein L7/L12 antigen and the gold colloid-labeled antibody.

(c) Preparation of Immunochromatographic Device

A sectional view of immunochromatographic device is shown in FIG. 2. Inaddition to the aforementioned labeled antibody-impregnated member 1 andmembrane carrier 2 for chromatographic development, 25 mm GF/DVA (filtermember 4 having a thickness of 776 μm and consisting of glass fibers,retention particle size 3.5 μm, GE Healthcare Bioscience) and 20 mmGF/AVA (filter member 7 having a thickness of 299 μm and consisting ofglass fibers, retention particle size 1.7 μm, GE Healthcare Bioscience)were adhered to each other as a member serving as both the member forsample addition and the member for removal of fat globules (third part),and filter paper as the member 5 for absorption was further prepared.After these members were adhered on a substrate 6 (thickness 254 μm,made of polystyrene, having adhesive for adhering the members), theywere cut in a width of 5 mm to prepare the immunochromatographic device.

Example 4 Evaluation of Bacteriolytic Activity by Immunochromatography 1

Measurement for cow's milk using the immunochromatographic device wasperformed as follows. Milk (300 μl) containing Streptococcus uberis at afinal concentration of 1×10⁵ (CFU/ml) was put into a microtube, 500 μlof a lysis treatment solution was added to the milk, and the treatmentwas performed at room temperature for 30 minutes. As the cow's milk,marketed milk for drinking was used. The aforementionedimmunochromatographic device was immersed into the above mixed solutionfrom the member 4 for sample addition, chromatographic development wasallowed by leaving the device standing at room temperature for 30minutes, and then for determining the presence or absence of capture ofthe complex of the ribosomal protein L7/L12 antigen and the goldcolloid-labeled antibody by the aforementioned part 3 for capturing,intensity of a reddish purple line that became more or less conspicuousin proportion to the capture amount was measured. The lysis ratiosobserved under various conditions are shown in Table 3.

TABLE 3 Condition Condition Condition Comparative 1 2 3 Example 3 Enzyme1 0.25 mg/ml 0.25 mg/ml 0.25 mg/ml 5 mg/ml Labiase Labiase LabiaseLysozyme Enzyme 2 None 0.75 mg/ml 0.75 mg/ml 4 mg/ml Lysozyme LysozymeAchromopeptidase Surfactant 1 (final 0.6% Triron 0.6% Triron 0.6% Triron0.2% Sodium N- concentration, wt/wt %) X-100 X-100 X-100 lauroylsalcosinate Surfactant 2 (final None None 0.6% Tween 20 0.2% Brij 58concentration, wt/wt %) Lysis ratio for S. uberis 35.0 48.0 56.1 1.5

It was revealed that, compared with the condition 1, in which onlylabiase was used, the lysis ratio was improved with the condition 2, inwhich lysozyme was used in combination, and the condition 3, in whichTween 20 (polyoxyethylene sorbitan fatty acid ester) was further addedas a surfactant. Further, when the condition described in Non-patentdocument 1 (Comparative Example 3) was retested, a low lysis ratio wasobserved.

Example 5 Evaluation of Bacteriolytic Activity by Immunochromatography 2

Addition amount of lysozyme used in combination with labiase wasevaluated by using the same method as that used in Example 4. Theresults are shown in Table 4. It was confirmed that addition ofappropriate amount of lysozyme improves lysis ratio.

TABLE 4 Compara- Condition Condition Condition tive 3 4 5 Example 9Enzyme 1 0.25 mg/ml Labiase Enzyme 2 Lysozyme Lysozyme Lysozyme Lysozyme0.75 mg/ml 1.25 mg/ml 3 mg/ml 6.25 mg/ml Surfactant 1 1% Triton X-100(final concen- tration, wt/wt %) Surfactant 2 1.3% Tween 20 (finalconcen- tration, wt/wt %) Lysis ratio for 56.1 62.2 67.3 15 (bad flow S.uberis of test liquid)

Example 6 Evaluation of Surfactant Concentration by Immunochromatography

Concentration of surfactants was evaluated by using the same method asthat used in Example 4. The results are shown in Table 5. It wasrevealed that if the addition amounts of Triton X-100 and Tween 20 areappropriate, the lysis ratio is further improved.

TABLE 5 Condi- Condi- Condi- Condi- Condi- Condi- ComparativeComparative tion 3 tion 6 tion 7 tion 8 tion 9 tion 10 Example 7 Example8 Enzyme 1 0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.25 mg/ml mg/ml mg/mlmg/ml mg/ml mg/ml mg/ml mg/ml Labiase Labiase Labiase Labiase LabiaseLabiase Labiase Labiase Enzyme 2 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml mg/ml Lysozyme LysozymeLysozyme Lysozyme Lysozyme Lysozyme Lysozyme Lysozyme Surfactant 1 0.6%Triton X-100 1% Triton X-100 1.3% Triton 1% Triton 2.5% Triton (finalconcen- X-100 X-100 X-100 tration, wt/wt %) Surfactant 2 0.6% 1% 1.3% 1%1.3% 1.3% 2.5% 2.5% (final concen- Tween 20 Tween 20 Tween 20 Tween 20Tween 20 Tween 20 Tween 20 Tween 20 tration, wt/wt %) Lysis ratio of56.1 70.4 69.8 60.9 62.6 83.5 44.6 38.4 S. uberis

Example 7 Detection Sensitivity for Live Cells Contained in Milk

Samples prepared by adding Streptococcus uberis at variousconcentrations to milk were treated with the lysis agents of theconditions 1 and 10, and measurement was carried out byimmunochromatography. A reddish purple line appeared was visuallyexamined (strongly positive ++, positive +, negative −).

TABLE 6 Number of bacteria CFU/mL Condition 1 Condition 10 1 × 10⁴ − + 2× 10⁴ − + 5 × 10⁴ + + 1 × 10⁵ + ++

Example 8 Improvement of Lysis Ratio with Additives

Lysis ratios obtainable with the lysis agents of the condition 9mentioned in Example 6 and further containing various additives wereevaluated. The results are shown in Table 7.

TABLE 7 Condition 9 Condition 11 Condition 12 Condition 13 Condition 14Condition 15 Condition 16 Enzyme 0.25 mg/ml Labiase, 0.75 mg/ml LysozymeSurfactant 1% Triton X-100, 1.3% Tween 20 Additive None 50 mM 50 mM 10mM 0.1 mM 1 mM 1 mM NaCl Guanidine Decanoic Monocaprylin EDTA EGTAthiocyanate acid Lysis ratio 62.6% 71.7% 85.4% 89.2% 91.2% 91.1% 92.4% *Monocaprylin: 1-monooctanoylglycerol, Tokyo Chemical Industry

INDUSTRIAL APPLICABILITY

The present invention can be utilized for quick diagnosis of mastitis oflivestock animals based on the immunochromatographic method, or thelike.

1. A method for lysing a Streptococcus bacterium contained in milk,which comprises: the step of mixing a lysis agent containing lysozyme,labiase, and a nonionic surfactant with the milk to lyse the bacteriumexisting in the milk; and the step of detecting a specific substancederived from the inside of the bacterium cell and released by lysis. 2.The lysis method according to claim 1, wherein the nonionic surfactantcomprises a polyoxyethylene alkyl phenyl ether and/or a polyoxyethylenesorbitan fatty acid ester.
 3. The lysis method according to claim 1,wherein, in the step of mixing the lysis agent with the milk to lyse thebacterium existing in the milk, final concentration of the nonionicsurfactant is not lower than 0.03% and not higher than 3%.
 4. The lysismethod according to claim 1, wherein, in the step of mixing the lysisagent with the milk to lyse the bacterium existing in the milk, finalconcentration of lysozyme is not lower than 0.5 mg/ml and not higherthan 200 mg/ml; and/or in the step of mixing the lysis agent with themilk to lyse the bacterium existing in the milk, final concentration oflabiase is not lower than 0.05 mg/ml and not higher than 20 mg/ml. 5.The detection method according to claim 1, which is performed by animmunochromatographic method.
 6. The detection method according to claim5, wherein the immunochromatographic method comprises (1) the step ofcontacting the milk containing the specific substance with a test striphaving a first part retaining a labeled first antibody directed to thespecific substance, or the specific substance that is labeled, a secondpart disposed downstream from the first part, on which a second antibodydirected to the specific substance is immobilized, and a third partdisposed upstream from the first part or the second part and havingvoids enabling removal of milk fat globules contained in the milk, atthe third part or a part existing upstream therefrom, and (2) the stepof flowing the milk up to the second part or a part existing downstreamtherefrom to obtain a detectable signal of the label at the second partor a part existing downstream therefrom.
 7. The detection methodaccording to claim 6, wherein the labeled first antibody directed to thespecific substance is retained in the first part.
 8. The detectionmethod according to claim 6, wherein the third part is constituted bytwo or more kinds of members having voids that can remove milk fatglobules of different particle sizes, respectively.
 9. The detectionmethod according to claim 8, wherein the third part is constituted by afirst member disposed downstream and a second member disposed upstream,and retention particle size of the second member is larger thanretention particle size of the first member.